Optical biosensor

ABSTRACT

The present invention relates to an optical biosensor comprising a porous matrix. In the specific case, reference is made to anodized porous alumina, on the surface of which the biological component specific for the analyte in question is immobilized, and to an optical-signal detector connected to said matrix. The present patent further relates to a biosensor having the porous matrix and the optical detector integrated in a single structure, in particular to biosensors with porous matrix other than porous alumina, for example porous silicon.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Divisional Application of U.S. application Ser. No. 10/858,370filed Jun. 2, 2004; the entire disclosure of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a porous-matrix optical biosensor. Inparticular, the present invention relates to an optical biosensor withporous matrix constituted by anodized porous alumina connected to adetector, preferably a photodiode, for detection of the signal. Thepresent invention moreover relates to a biosensor having an opticaldetector integrated with a porous matrix other than porous alumina, suchas for example porous silicon.

A biosensor is a device capable of detecting a chemical or biochemicalvariable (analyte) by means of a biological component (biomediator),which, being immobilized on a matrix/substrate, functions as interfacewith a transducer. The transducer, which is constituted by thesensitized matrix and by a detector, is capable of transforming thechemico-physical signal deriving from the interaction between thebiomediator and the analyte into a measurable physical (i.e.,electrical) signal, which depends upon the variable analysed. Thedetector is able to measure the physical signal both in purelyqualitative terms and in quantitative terms.

An optical biosensor is a device capable of measuring theluminescence—whether chemiluminescence or bioluminescence—emitted duringthe interaction between the biomediator and its corresponding biologicalvariable. Said interaction entails, in fact, occurrence of a chemicalreaction which brings about the passage of one of the species involvedin the reaction into an electronically excited state. Decay of saidspecies from the excited state to the fundamental state brings aboutemission of photons (hv), the measurement of which supplies anindication not only of the presence but also of the amount of theanalyte being measured.

The essential characteristics of biosensors are the sensitivity and theselectivity that the biological component is able to provide, inconjunction with the simplicity of use and the versatility that derivesfrom the method of transduction chosen, which is usually compatible withspecifications of low-cost miniaturizability.

The biomediators or biological systems used may be enzymes (e.g.,luciferase), antibodies, biological membranes, bacteria of a wild strainor genetically modified bacteria (e.g., natural or recombinantbioluminescent bacteria), cells, animal or vegetable tissues; theseinteract directly or indirectly with the analyte to be determined andare responsible for the specificity of the sensor. The biomediatorinteracting with the analyte brings about a variation in one or morechemico-physical parameters of the species involved, giving, forexample, rise to a chemiluminescent or bioluminescent reaction withcorresponding emission of photons (hv).

The substrates used for immobilization of the biomediator can beconstituted by various materials. Amongst the currently used ones therecan mentioned silica gel, agarose, polymeric compounds such as, forexample, polystyrene or polyacrylates, natural fibres such as silk, orelse glass (micro)spheres.

The areas of application of biosensors are very wide and range from themedico-diagnostic sector to the environmental and foodstuff sectors.

In the foodstuff sector, biosensors can be used for determining chemicalsubstances that may function as indicators, for example, of themicrobial pollution present in a foodstuff or of the deterioration ofthe latter, for example caused by processes of oxidation. It is moreoverpossible to detect traces of contaminating chemical compounds, toxins,or else additives, preservatives, etc.

Also the applications in the environmental sector are extremely numerousfor determining the presence of pesticides, hydrocarbons, and toxicgases. In many cases, on account of the need to detect levels ofconcentration that fall below the range of detection of the biosensor,the latter has been coupled, in the case of electrical transduction, toelectronic amplifiers.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an optical biosensorhaving a structure such as to facilitate the contacts between thesensitized porous matrix and the detector—i.e., to facilitate thefunctionality of the transducer—with consequent advantages in thedetection of the signal generated by the transducer as a function of theinteraction between the biomediator and the analyte.

According to the invention, the above purpose is achieved thanks to thesolution recalled specifically in the ensuing claims, which areunderstood as forming an integral part of the present description.

In the currently preferred embodiment, the invention relates to anoptical biosensor having as matrix (substrate), a material with porousstructure, preferably anodized porous alumina, on which the biomediatoris immobilized.

BRIEF DESCRIPTION OF THE DRAWINGS

Further characteristics and advantages of the biosensor according to thepresent invention will emerge clearly from the ensuing detaileddescription, provided purely by way of non-limiting example, withreference to the annexed drawings, in which:

FIG. 1 represents an optical biosensor according to the invention;

FIG. 2 reproduces two photographs obtained with the scanning electronicmicroscope of a cross section and a front section of a matrix ofanodized porous alumina;

FIG. 3 illustrates the structure of an optical biosensor according tothe invention;

FIG. 4 represents an optical biosensor according to the inventioncoupled to a photodiode;

FIG. 5 illustrates an optical biosensor having a porous matrixconsisting of porous silicon; and

FIG. 6 reproduces a further embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIG. 1, number 1 designates, as a whole, the opticalbiosensor. The biosensor 1 comprises a matrix or supporting structure 2made of anodized porous alumina, inside the pores 2 a of which there isimmobilized the biomediator 3, which is able to react with the analyte 5contained in the solution to be analysed 6. The porous matrix 2 isconnected to, and preferably integrated with, an optical detector 4,capable of measuring the signal—emission of photons hv—generated by thereaction between the biomediator 3 and the analyte 5. The photonsemitted during the reaction between the biomediator and the analyte arecorrelated, when the biomediator is not in conditions of saturation, tothe amount of analyte present in the solution 6.

The innovative aspect of the present patent is represented by the use ofa porous matrix 2 consisting of porous alumina obtained via a process ofanodization of a film of high-purity aluminium or of a film of aluminiumadhering to substrates such as glass, quartz, silicon, tungsten, etc.

The peculiar characteristics of anodized porous alumina are outlined inwhat follows. In the first place, the regularity of the pores bestowsupon the material particular optical properties; in fact, the structuralperiodicity of the aforesaid material enables alternation of means withdifferent dielectric constants, producing a photonic band gap that doesnot enable propagation of light radiation in a specific band ofwavelengths and in certain directions, with consequent narrowing of theemission lobe of the outcoming light. In addition, the porous surfacebrings about a considerable increase in the area of possible contact.The latter aspect favours substantially the process of immobilization ofthe biomediator, which can reach higher concentrations per unit area ascompared to the use of a compact smooth structure.

The dimensions and number of pores can be controlled by varying theconditions of anodization of metallic aluminium.

The choice of metallic aluminium as a starting material presents a majoradvantage: it can be deposited on any surface using evaporationtechniques and be subsequently anodized. In this way, it is possible todeposit a layer of aluminium—subsequently subjected toanodization—directly on an optical detector (for example, a photodiode),so guaranteeing a further miniaturization of the biosensor.

The choice of porous alumina as a matrix further enables the use ofphotolithographic techniques followed by chemical etching, which enablethe generation of any three-dimensional or two-dimensional structure ofthe matrix.

The subsequent opening of the pores of the alumina matrix enablestreatment of the matrix as a true membrane and facilitates formation ofthe electrical contacts in the transducer.

FIG. 2 illustrates, purely by way of example, a portion of a film ofporous alumina obtained via anodic oxidation of an aluminium film. Asmay be noted, the layer of alumina is formed by a series of adjacentcells of a substantially hexagonal shape, each having a straight centralhole which constitutes a hole substantially perpendicular to the surfaceof the underlayer (FIG. 2 a).

As per the known art, the film of porous alumina can be developed withcontrolled morphology by appropriately choosing the electrolyte and thephysical, chemical and electrochemical parameters of the process.

Briefly, the first step for integration of a photodiode to the biosensoris the deposition of an aluminium layer on an underlayer, the latterbeing, for example, made of silicon on which there have previously beeninserted nanoclusters made of gold. The preferred techniques fordeposition of the layer of aluminium are thermal evaporation via e-beam,and cathodic sputtering. The step of deposition of the aluminium layeris followed by a step of anodization of the layer itself. The process ofanodization of the layer can be carried out using different electrolyticsolutions according to the size and distance of the pores that are to beobtained. In order to obtain a highly regular structure, of the sametype as the one represented in FIG. 2, it becomes necessary to carry outsubsequent anodization processes, and, in particular, at least:

i) a first anodization;

ii) a step of reduction, via chemical etching, of the irregular film ofalumina by means of acidic solutions; and

iii) a second anodization of the part of the film of alumina noteliminated during the step of chemical etching.

The etching step referred to in point ii) is important for defining onthe residual part of alumina preferential areas of growth of the aluminaitself in the second anodization step.

If the operations of etching ii) and anodization iii) are carried out anumber of times, the structure improves until it becomes very uniform,as highlighted schematically in FIG. 2, where the film of alumina isregular.

The regular structure of porous alumina can be developed with controlledmorphology by appropriately choosing the electrolyte and the physical,chemical and electrochemical parameters of the process: in acidicelectrolytes (such as phosphoric acid, oxalic acid and sulphuric acidwith concentrations of 0.2-1.2M) and in adequate process conditions(voltage of 40-200 V, current density of 5-10 mA/cm², stirring, andtemperature of 0-4° C.), it is possible to obtain porous filmspresenting a high level of regularity. The diameter of the pores and thedepth of the film may be varied; typically, the diameter is 50-500 nmand the depth 1-200 μm.

In the present invention, as a signal detector, and hence in the case inpoint as optical-signal detector, any system sensitive to light, such asa photodiode, may be used, where by the term “photodiode” is meant aphotodiode formed by two or more sections.

Alternatively, as detectors it is possible to use any light-sensitivemeans integrated with the porous matrix. By way of example, amongst thetechniques that enable this integration between the optical detector,and the porous matrix the following may be mentioned:

-   -   the nanopatterning technique, which envisages a process of        deposition of a metal on the bottom portion of the porous matrix        for a better adhesion of the photodiode to the matrix, as        illustrated schematically in FIG. 4; and    -   the integration technique, which envisages deposition on a        silicon substrate of gold nanoclusters and then of the porous        matrix (FIG. 3).

In the embodiment illustrated in FIG. 4, the biosensor, which isdesignated, as a whole, by the reference number 1, consists of a matrixof anodized porous alumina 2, inside the pores 2 a of which thebiomediators 3 specific for the analyte 5 contained in the solution tobe analysed 6, are immobilized. The detector 14 of the light signalemitted by the interaction between the biomediator 3 and the analyte 5is constituted by a photodiode 14 a and a metal layer 14 b adhering onthe bottom surface 2 b of the porous matrix for the purpose of improvingthe transmission of photons hv between the matrix and the photodiode.

FIG. 3 represents another embodiment of the present invention. Thematrix 2 of anodized porous alumina—inside the pores 2 a of which thebiomediator 3 is immobilized—is in contact with the detector 24constituted by a series of metal nanoclusters 24 b, preferably goldnanoclusters, deposited on a silicon substrate 24 a so that the photonshv emitted during the biomediator-analyte interaction will be absorbedby the detector formed by the metal-silicon junction and will bedetected by measuring the electrical potential at the junction.

Other optical detectors that can advantageously be used for theembodiment of the present invention may be represented by polymericphotodiodes, such as, for example, LEP (Light-Emitting Polymer) opticalsensors or OLED (Organic Light-Emitting Diode) optical sensors. The useof these polymeric photodiodes presents the major advantage of employingflexible structures with high biocompatibility. An advantageous exampleof a possible application of this particular embodiment of the presentinvention is provided by the integration of this biosensor in adiagnostic instrument such as an endoscope; the endoscope presents thebiosensor throughout its length for instantaneous monitoring of theanalyte in question along an extensive stretch of the organ beingexamined.

An alternative embodiment of the present invention envisages thepossibility of integrating the optical detector with biosensors formedby a porous matrix other than porous alumina; by way of example,reference will be made to porous silicon.

The porosity of the silicon matrix can be varied according to thebiological species (biomediators) and hence to the analytes that are tobe detected. With reference to FIG. 5, the porous silicon 2 is coated byelectrochemical deposition with a continuous metal layer 54 a (forexample, gold) so as to function itself as a photodiode 54, there beingcreated a Schottky junction. The internal walls of the pores 2 a ofporous silicon, coated with metal 54 a, also function as substrate forthe biomediator 3, which is immobilized thereon. In order to immobilizethe mediator the techniques described hereinafter are used.

When the reaction between the biomediator and the analyte brings aboutemission of photons, these are immediately absorbed by the photodiode 54constituted by the metal-silicon junction and are detected by measuringthe electrical potential that is set up between the silicon and themetal.

The main advantage of the above embodiment of the present invention isprovided by the complete integration of the porous matrix sensitizedwith the biomediator and of the optical sensor, with evident advantagesin terms of design, reduction of the technological process steps and ofthe costs of the devices themselves.

In a particular embodiment represented schematically in FIG. 6, it ispossible to provide an optical biosensor according to the presentinvention having the porous matrix divided into two or more sections, sothat each section will be sensitized with a different biomediatorcapable of detecting one specific analyte and the biosensor as a wholeshall be capable of detecting simultaneously two or more analytes ofinterest, thus obtaining a so-called “lab-on-chip”. The individualsections are separate from one another, and, through exploitation of asystem of injectors and reservoirs, the efficiency of the biomediatorsis guaranteed.

With reference to FIG. 6, the porous matrix 2 of the biosensor 1 isdivided into four sections 22 a, 22 b, 22 c and 22 d sensitized withfour different biomediators which are specific for different analytes.This division is obtained by means of barriers 7 a and 7 b, which enablethe four environments 6 a, 6 b, 6 c, 6 d for the biomediator-analytereaction to be kept separate. Also the detector is divided according tothe partitions of the porous matrix so that the signals of thebiomediator-analyte interactions of the four reaction environments maybe processed separately, but simultaneously, by the detectors 64 a, 64b, 64 c and 64 d, and a multiparametric analysis can be conducted.

The immobilization of the biomediator (whether this be an enzyme or acomplex biological organism) on the porous matrix can be achieved by awide range of techniques. By way of non-limiting example, the followingmay be mentioned:

-   -   formation of non-covalent bonds (e.g., hydrogen bonds, Van der        Waals bonds) between the biomediator and the porous matrix        possibly functionalized in an appropriate manner;    -   micro-encapsulation through the use of membranes of porous        alumina capable of entrapping the biomediator;    -   formation of covalent bonds between the biomediator and the        porous matrix, optionally appropriately functionalized; and    -   cross-bonding with a bifunctional chemical compound capable of        setting up a chemical bond between the matrix on the one hand        and the biomediator on the other (this method can be used in        concomitance with other immobilization techniques, such as        absorption and micro-encapsulation).

The techniques that envisage the use of non-covalent bonds forimmobilization of the biomediator to the matrix are preferable, in sofar as they do not require any chemical modification of the biomediator.In this case, the surface of the porous alumina is preferablyimpregnated with any compound capable of increasing adhesion of thebiomediator to the surface itself. An example of one of these compoundsis given by a polylysine peptide, which, by being adsorbed on thehydrophilic surface of the alumina, is then capable of “co-ordinating”with the biomediator exploiting the presence of —NH₂ groups on its sidechain and hence giving rise to hydrogen bonds and/or of Van der Waalsbonds with the hydrophilic groups of the biomediator. A second exampleof a compound capable of increasing adhesion of the biomediator to thealumina matrix is polyprenyl phosphate; the phosphate group functions asan anchor capable of being adsorbed on the alumina, and the prenylenictail—by rendering the alumina surface more hydrophobic—will favour theformation of non-covalent bonds between the matrix thus functionalizedand the biomediator.

Of course, without prejudice to the principle of the invention, thedetails of construction and the embodiments may vary respect to what isdescribed and illustrated herein, without thereby departing from thescope of the invention.

1. A biosensor comprising a porous matrix made of porous silicon, abiomediator immobilized on said matrix; and an optical-signal detectorconnected to said matrix; wherein said porous matrix and said detectorare integrated in a structure comprising: a metal layer deposited onsaid porous matrix, and said biomediator is immobilized on said metallayer, wherein the optical-signal detector is constituted by thecombination of said metal layer and said porous silicon matrix.
 2. Thebiosensor according to claim 1, wherein the metal layer is a continuousmetal layer.
 3. The biosensor according to claim 1, wherein the metallayer is a gold metal layer.
 4. The biosensor according to claim 1,wherein the metal layer is an electrochemically deposited metal layer.5. The biosensor according to claim 1, wherein said biomediator isimmobilized on said metal layer by non-covalent bonds between saidbiomediator and said metal layer; micro-encapsulation of the biomediatoron said metal layer; covalent bonds between said biomediator and saidmetal layer; and/or cross-bonding with a bifunctional chemical compoundcapable of forming a bond between said metal layer and said biomediator.6. The biosensor according to claim 5, wherein said biomediator isimmobilized on said metal layer by the bifunctional chemical compound.